RNAi-mediated knockdown of exportin 1 negatively affected ovary development, survival and maize mosaic virus accumulation in its insect vector Peregrinus maidis.

Exportin 1 (XPO1) is the major karyopherin-ß nuclear receptor mediating the nuclear export of hundreds of proteins and some classes of RNA and regulates several critical processes in the cell, including cell-cycle progression, transcription and translation. Viruses have co-opted XPO1 to promote nucleocytoplasmic transport of viral proteins and RNA. Maize mosaic virus (MMV) is a plant-infecting rhabdovirus transmitted in a circulative propagative manner by the corn planthopper, Peregrinus maidis. MMV replicates in the nucleus of plant and insect hosts, and it remains unknown whether MMV co-opts P. maidis XPO1 (PmXPO1) to complete its life cycle. Because XPO1 plays multiple regulatory roles in cell functions and virus infection, we hypothesized that RNAi-mediated silencing of XPO1 would negatively affect MMV accumulation and insect physiology. Although PmXPO1 expression was not modulated during MMV infection, PmXPO1 knockdown negatively affected MMV accumulation in P. maidis at 12 and 15 days after microinjection. Likewise, PmXPO1 knockdown negatively affected P. maidis survival and reproduction. PmXPO1 exhibited tissue-specific expression patterns with higher expression in the ovaries compared with the guts of adult females. Survival rate was significantly lower for PmXPO1 knockdown females, compared with controls, but no effect was observed for males. PmXPO1 knockdown experiments revealed a role for PmXPO1 in ovary function and egg production. Oviposition and egg hatch on plants were dramatically reduced in females treated with dsRNA PmXPO1. These results suggest that PmXPO1 is a positive regulator of P. maidis reproduction and that it plays a proviral role in the insect vector supporting MMV infection.

Zika Virus Infection in the Ovary Induces a Continuously Elevated Progesterone Level and Compromises Conception in Interferon Alpha/Beta Receptor-Deficient Mice.

Zika virus (ZIKV) belongs to mosquito-borne flaviviruses. Unlike other members in the family, ZIKV can be sexually transmitted, and the female genital tracts are susceptible to ZIKV. However, the impact of ZIKV infection on nonpregnant female reproductive health is not understood. In this study, we investigated the effects of ZIKV infection on the ovary by using nonpregnant female interferon α/ß receptor-deficient (Ifnar1-/-) mice. The results showed that the ovary supported ZIKV replication, and the granulosa and theca cells of antral follicles were susceptible. ZIKV replication in situ significantly reduced the numbers of antral follicles, aggravated follicular atresia, and disrupted folliculogenesis. Notably, ZIKV replication in the ovary caused disordered ovarian steroidogenesis manifested by decreased expression of key enzymes linked to sex hormone synthesis, including the cytochrome P450 17A1 (CYP17A1) and aromatase (CYP19A1). Further, we observed that ZIKV infection disrupted the estrous cycle and thus prolonged the time to conceive. More importantly, although ZIKV RNA could not be detected at 3 months postinfection, damaged ovarian structure and dysfunction were also observed. Taken together, our study demonstrates that ZIKV infection in nonpregnant female mice cause ovarian damage and dysfunction, even long after ZIKV clearance. These data provide important information to understand the effects of ZIKV infection in female reproductive tissues and basic evidence for further studies. IMPORTANCE Zika virus (ZIKV), a flavivirus, is primarily transmitted by mosquito bites. But it can also be transmitted vertically and sexually. Although ZIKV-associated Guillain-Barré syndrome and microcephaly have drawn great attention, there have been few studies on the potential effects of ZIKV on the genital tract of nonpregnant females. This study investigated the effects of ZIKV on the ovaries in mice. We found that ZIKV replicated in the ovary and the granulosa and theca cells of antral follicles were susceptible. ZIKV replication in situ significantly damaged ovarian structure and function and disrupted folliculogenesis. Notably, ZIKV infection further disrupted the estrous cycle and prolonged the time to conceive in mice by causing disordered ovarian steroidogenesis. These effects were observed in both the acute phase and the recovery phase after viral elimination. Overall, the new findings provide important additions to make out the potential adverse impacts of ZIKV on reproductive health in females.

Co-expression of the SARS-CoV-2 entry molecules ACE2 and TMPRSS2 in human ovaries: Identification of cell types and trends with age.

The high rate of SARS-CoV-2 infection poses a serious threat to public health. Previous studies have suggested that SARS-CoV-2 can infect human ovary, the core organ of the female reproductive system. However, it remains unclear which type of ovarian cells are easily infected by SARS-CoV-2 and whether ovarian infectivity differs from puberty to menopause. In this study, public datasets containing bulk and single-cell RNA-Seq data derived from ovarian tissues were analyzed to demonstrate the mRNA expression and protein distribution of the two key entry receptors for SARS-CoV-2-angiotensin-converting enzyme 2 (ACE2) and type II transmembrane serine protease (TMPRSS2). Furthermore, an immunohistochemical study of ACE2 and TMPRSS2 in human ovaries of different ages was conducted. Differentially expressed gene (DEG) analysis of ovaries of different ages and with varying ovarian reserves was conducted to explore the potential functions of ACE2 and TMPRSS2 in the ovary. The analysis of the public datasets indicated that the co-expression of ACE2 and TMPRSS2 was observed mostly in oocytes and partially in granulosa cells. However, no marked difference was observed in ACE2 or TMPRSS2 expression between young and old ovaries and ovaries with low and high reserves. Correspondingly, ACE2 and TMPRSS2 were detected in the human ovarian cortex and medulla, especially in oocytes of different stages, with no observed variations in their expression level in ovaries of different ages, which was consistent with the results of bioinformatic analyses. Remarkably, DEG analysis showed that a series of viral infection-related pathways were more enriched in ACE2-positive ovarian cells than in ACE2-negative ovarian cells, suggesting that SARS-CoV-2 may potentially target specific ovarian cells and affect ovarian function. However, further fundamental and clinical research is still needed to monitor the process of SARS-CoV-2 entry into ovarian cells and the long-term effects of SARS-CoV-2 infection on the ovarian function in recovered females.

Route of Zika virus infection in Aedes aegypti by transmission electron microscopy.

BACKGROUND: Zika fever has been a global health security threat, especially in the tropical and subtropical regions where most of the cases occur. The disease is caused by Zika virus (ZIKV), which belongs to the family Flaviviridae, genus Flavivirus. The virus is transmitted by Aedes mosquitoes, mostly by Aedes aegypti, during its blood meal. In this study we present a descriptive analysis, by transmission electron microscopy (TEM), of ZIKV infection in A. aegypti elected tissues at the 3rd day of infection. ZIKV vertical transmission experiments by oral infection were conducted to explore an offspring of natural infection. RESULTS: Gut and ovary tissues harbored a higher number of viral particles. The ZIKV genome was also detected, by RT-qPCR technique, in the organism of orally infected female mosquitoes and in their eggs laid. CONCLUSIONS: The data obtained suggest that the ovary is an organ susceptible to be infected with ZIKV and that virus can be transmitted from mother to a fraction of the progeny.

Ovarian HBV replication following ovulation induction in female hepatitis B carriers undergoing IVF treatment: A prospective observational study.

Hepatitis B virus (HBV) can be found in ovarian tissues. This study compared HBV DNA levels in follicular fluid collected during oocyte retrieval with paired serum samples in HBV carriers after ovarian stimulation during IVF treatment for infertility. Sixty-four HBV carrier women referred to the Assisted Reproductive Units of two Hong Kong hospitals were recruited. At oocyte retrieval, the follicular fluid aspirated from the first follicle was collected for study. In 22 women, the first follicular fluid sample from both ovaries was similarly collected and studied. These women were also tested for liver function test and HBeAg. In 28 (43.8%) women, HBV DNA was detected in follicular fluid and the level correlated with serum levels (Spearman's correlation P < .001). There was concordant detection of HBV DNA in both ovaries, and the levels were significantly correlated (Spearman's correlation P = .029). In 40% of women with FF HBV DNA, the follicular fluid:serum ratio was >1.0, suggesting stimulation of HBV replication. These women also had significantly different liver function test results. Increased HBV replication exists in 40% of women with HBV DNA detected in follicular undergoing ovarian stimulation during IVF treatment.

Characteristics of laboratory indexes in COVID-19 patients with non-severe symptoms in Hefei City, China: diagnostic value in organ injuries.

This study compared the laboratory indexes in 40 non-severe COVID-19 patients with those in 57 healthy controls. In the peripheral blood system of non-severe symptom COVID-19 patients, lymphocytes, eosinophils, basophils, total procollagen type 1 amino-terminal propeptide, osteocalcin N-terminal, thyroid-stimulating hormone, growth hormone, and insulin-like growth factor-binding protein 3 significantly decreased, and total protein, albumin, alanine transaminase, alkaline phosphatase, γ-glutamyl transferase, activated partial thromboplastin time, prothrombin time, fibrinogen, D-dimer, fibrinogen degradation products, human epididymal protein 4, serum ferritin, and C-reactive protein were elevated. SARS-CoV-2 infection can affect hematopoiesis, hemostasis, coagulation, fibrinolysis, bone metabolism, thyroid, parathyroid glands, the liver, and the reproductive system.

Vector development and vitellogenin determine the transovarial transmission of begomoviruses.

The majority of plant viruses are transmitted by insect vectors between hosts, and transovarial transmission of viruses from vector parents to offspring has great significance to their epidemiology. Begomoviruses are transmitted by the whitefly Bemisia tabaci in a circulative manner and are maintained through a plant-insect-plant cycle. Other routes of begomovirus transmission are not clearly known. Here, we report that transovarial transmission from female whiteflies to offspring often happens for one begomovirus, Tomato yellow leaf curl virus (TYLCV), and may have contributed significantly to its global spread. We found that TYLCV entry of the reproductive organ of its vector mainly depended on the developmental stage of the whitefly ovary, and the transovarial transmission of TYLCV to offspring increased with whitefly adult age. The specific interaction between virus coat protein (CP) and whitefly vitellogenin (Vg) was vital for virus entry into whitefly ovary. When knocking down the expression of Vg, the entry of TYLCV into ovary was inhibited and the transovarial transmission efficiency decreased. In contrast, another begomovirus, Papaya leaf curl China virus (PaLCuCNV), CP did not interact with whitefly Vg, and PaLCuCNV could not be transovarially transmitted by whiteflies. We further showed that TYLCV could be maintained for at least two generations in the absence of virus-infected plants, and the adult progenies were able to infect healthy plants in both the laboratory and field. This study reports the transovarial transmission mechanism of begomoviruses, and it may help to explain the evolution and global spread of some begomoviruses.

SARS-CoV-2 and the next generations: which impact on reproductive tissues?

Coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is a severe global pandemic, affecting mostly the respiratory system. Understandably, attention is also being directed towards the urogenital tract. In this work, expression patterns of various host molecules possibly involved in viral entry and replication were investigated in human female and male reproductive systems by inquiring online repositories, including the Human Protein Atlas, GTEx, FANTOM5. Our findings highlight that male reproductive tissues could be targeted by SARS-CoV-2, particularly the testis since it co-expresses the receptor (ACE2) and the protease (TMPRSS) needed for viral entry. We hypothesized that SARS-CoV-2 infection could have repercussions on the fertility status of male individuals Potential infectivity of SARS-CoV-2 in reproductive tissues should be considered in reproductive medicine and management of in vitro fertilization in present and future generations.

Ebola Virus Localization in the Macaque Reproductive Tract during Acute Ebola Virus Disease.

Sexual transmission of Ebola virus (EBOV) has been demonstrated more than a year after recovery from the acute phase of Ebola virus disease (EVD). The mechanisms underlying EBOV persistence and sexual transmission are not currently understood. Using the acute macaque model of EVD, we hypothesized EBOV would infect the reproductive tissues and sought to localize the infection in these tissues using immunohistochemistry and transmission electron microscopy. In four female and eight male macaques that succumbed to EVD between 6 and 9 days after EBOV challenge, we demonstrate widespread EBOV infection of the interstitial tissues and endothelium in the ovary, uterus, testis, seminal vesicle, epididymis, and prostate gland, with minimal associated tissue immune response or organ pathology. Given the widespread involvement of EBOV in the reproductive tracts of both male and female macaques, it is reasonable to surmise that our understanding of the mechanisms underlying sexual transmission of EVD and persistence of EBOV in immune-privileged sites would be facilitated by the development of a nonhuman primate model in which the macaques survived past the acute stage into convalescence.

New cell lines for efficient propagation of koi herpesvirus and infectious salmon anaemia virus.

The production of piscine viruses, in particular of koi herpesvirus (KHV, CyHV-3) and infectious salmon anaemia virus (ISAV), is still challenging due to the limited susceptibility of available cell lines to these viruses. A number of cell lines from different fish species were compared to standard diagnostic cell lines for KHV and ISAV regarding their capability to exhibit a cytopathic effect (CPE) and to accumulate virus. Two cell lines, so far undescribed, appeared to be useful for diagnostic purposes. Fr994, a cell line derived from ovaries of rainbow trout (Oncorhynchus mykiss), produced constantly high ISA virus (ISAV) titres and developed a pronounced CPE even at high cell passage numbers, while standard cell lines are reported to gradually loose these properties upon propagation. Another cell line isolated from the head kidney of common carp (Cyprinus carpio), KoK, showed a KHV induced CPE earlier than the standard cell line used for diagnostics. A third cell line, named Fin-4, established from the fin epithelium of rainbow trout did not promote efficient replication of tested viruses, but showed antigen sampling properties and might be useful as an in vitro model for virus uptake or phagocytosis.

Comprehensive Profiling of Lysine Acetylome in Baculovirus Infected Silkworm (Bombyx mori) Cells.

Bombyx mori is one of the key lepidopteran model species, and is economically important for silk production and proteinaceous drug expression. Baculovirus and insect host are important natural biological models for studying host-pathogen interactions. The impact of Bombyx mori nucleopolyhedrovirus (BmNPV) infection on the proteome and acetylome of Bombyx mori ovarian (BmN) cells are explored to facilitate a better understanding of infection-driven interactions between BmNPV and host in vitro. The proteome and acetylome are profiled through six-plex Tandem mass tag (TMT) labeling-based quantitative proteomics. A total of 4194 host proteins are quantified, of which 33 are upregulated and 47 are downregulated in BmN cells at 36 h post-infection. Based on the proteome, quantifiable differential Kac proteins are identified and functionally annotated to gene expression regulation, energy metabolism, substance synthesis, and metabolism after BmNPV infection. Altogether, 644 Kac sites in 431 host proteins and 39 Kac sites in 22 viral proteins are identified and quantified in infected BmN cells. Our study demonstrates that BmNPV infection globally impacts the proteome and acetylome of BmN cells. The viral proteins are also acetylated by the host acetyltransferase. Protein acetylation is essential for cellular self-regulation and response to virus infection. This study provides new insights for understanding the host-virus interaction mechanisms, and the role of acetylation in BmN cellular response to viral infection.

Presence of Penaeus monodon densovirus in the ovary of chronically infected P. monodon subadults.

Shrimp infected with Penaeus monodon densovirus (PmoDNV) usually display no specific gross signs, but heavy infections can kill postlarvae and retard juvenile growth. In the present study, samples of hepatopancreas, feces, gonads and hemolymph were isolated from male and female P. monodon subadults chronically infected by PmoDNV. Each sample of hepatopancreas and gonad was divided into 2 parts: one for PmoDNV detection by polymerase chain reaction (PCR), and the other for routine histology and immunohistochemistry. The frequency of positive findings via PCR assays was 92% in the hepatopancreas, 57% in feces, 50% in ovary, 35% in hemolymph and 0% in the testis. Using the densitometric value (DV) of the specific band for PmoDNV relative to that of the ß-actin gene as an index of the viral load in the samples, no significant differences were observed among sample types and sexes. Hematoxylin-eosin staining of infected hepatopancreas revealed typical PmoDNV inclusions in the nuclei of infected cells. The ovaries with high DV (>1) contained various types of inclusions along the row of the follicular cells or possibly in the connective tissue cells surrounding the oocytes. Using immunohistochemistry with specific probes to detect PmoDNV proteins, a positive reaction was observed in viral inclusions found in infected hepatopancreas and in ovaries with high DV, specifically in the ovarian capsule, hemolymph, oocytes and nuclear inclusions. These results suggest that the localization of PmoDNV in P. monodon is not confined to the hepatopancreas, but rather that the virus can also occur in the ovary; hence, trans-ovarian, vertical transmission of the virus is highly possible.

Natural Killer Cells and Innate Interferon Gamma Participate in the Host Defense against Respiratory Vaccinia Virus Infection.

UNLABELLED: In establishing a respiratory infection, vaccinia virus (VACV) initially replicates in airway epithelial cells before spreading to secondary sites of infection, mainly the draining lymph nodes, spleen, gastrointestinal tract, and reproductive organs. We recently reported that interferon gamma (IFN-γ) produced by CD8 T cells ultimately controls this disseminated infection, but the relative contribution of IFN-γ early in infection is unknown. Investigating the role of innate immune cells, we found that the frequency of natural killer (NK) cells in the lung increased dramatically between days 1 and 4 postinfection with VACV. Lung NK cells displayed an activated cell surface phenotype and were the primary source of IFN-γ prior to the arrival of CD8 T cells. In the presence of an intact CD8 T cell compartment, depletion of NK cells resulted in increased lung viral load at the time of peak disease severity but had no effect on eventual viral clearance, disease symptoms, or survival. In sharp contrast, RAG(-/-) mice devoid of T cells failed to control VACV and succumbed to infection despite a marked increase in NK cells in the lung. Supporting an innate immune role for NK cell-derived IFN-γ, we found that NK cell-depleted or IFN-γ-depleted RAG(-/-) mice displayed increased lung VACV titers and dissemination to ovaries and a significantly shorter mean time to death compared to untreated NK cell-competent RAG(-/-) controls. Together, these findings demonstrate a role for IFN-γ in aspects of both the innate and adaptive immune response to VACV and highlight the importance of NK cells in T cell-independent control of VACV in the respiratory tract. IMPORTANCE: Herein, we provide the first systematic evaluation of natural killer (NK) cell function in the lung after infection with vaccinia virus, a member of the Poxviridae family. The respiratory tract is an important mucosal site for entry of many human pathogens, including poxviruses, but precisely how our immune system defends the lung against these invaders remains unclear. Natural killer cells are a type of cytotoxic lymphocyte and part of our innate immune system. In recent years, NK cells have received increasing levels of attention following the discovery that different tissues contain specific subsets of NK cells with distinctive phenotypes and function. They are abundant in the lung, but their role in defense against respiratory viruses is poorly understood. What this study demonstrates is that NK cells are recruited, activated, and contribute to protection of the lung during a severe respiratory infection with vaccinia virus.

Sequence-independent characterization of viruses based on the pattern of viral small RNAs produced by the host.

Virus surveillance in vector insects is potentially of great benefit to public health. Large-scale sequencing of small and long RNAs has previously been used to detect viruses, but without any formal comparison of different strategies. Furthermore, the identification of viral sequences largely depends on similarity searches against reference databases. Here, we developed a sequence-independent strategy based on virus-derived small RNAs produced by the host response, such as the RNA interference pathway. In insects, we compared sequences of small and long RNAs, demonstrating that viral sequences are enriched in the small RNA fraction. We also noted that the small RNA size profile is a unique signature for each virus and can be used to identify novel viral sequences without known relatives in reference databases. Using this strategy, we characterized six novel viruses in the viromes of laboratory fruit flies and wild populations of two insect vectors: mosquitoes and sandflies. We also show that the small RNA profile could be used to infer viral tropism for ovaries among other aspects of virus biology. Additionally, our results suggest that virus detection utilizing small RNAs can also be applied to vertebrates, although not as efficiently as to plants and insects.

Spring viraemia of carp virus enters grass carp ovary cells via clathrin-mediated endocytosis and macropinocytosis.

Spring viraemia of carp virus (SVCV) is the causative pathogen of the outbreaks of an acute haemorrhagic and contagious viraemia responsible for the significant mortality in several cyprinid species. However, the endocytic pathway(s) and their regulatory molecules have not been characterized for SVCV. Here, using a combination of specific pharmacological inhibitors, transmission electron microscopy, immunofluorescence microscopy and real-time quantitative PCR, we found that SVCV entered grass carp ovary cells via clathrin-mediated endocytosis and macropinocytosis in a low-pH-dependent manner. We also discovered that dynamin II, actin microfilaments and microtubules were essential for SVCV internalization. Moreover, we found that the P21-activated kinase 1 inhibitor IPA-3 and the protein kinase C inhibitor rottlerin could block SVCV cell entry and replication, while phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 could promote SVCV infection. Results presented in this study provide helpful insight into revealing the initial steps of SVCV infection, and they may facilitate the development of therapeutic interventions.

Wolbachia uses a host microRNA to regulate transcripts of a methyltransferase, contributing to dengue virus inhibition in Aedes aegypti.

The endosymbiont Wolbachia is common among insects and known for the reproductive manipulations it exerts on hosts as well as inhibition of virus replication in their hosts. Recently, we showed that Wolbachia uses host microRNAs to manipulate host gene expression for its efficient maintenance in the dengue mosquito vector, Aedes aegypti. Cytosine methylation is mediated by a group of proteins called DNA (cytosine-5) methyltransferases, which are structurally and functionally conserved from prokaryotes to eukaryotes. The biological functions of cytosine methylation include host defense, genome stability, gene regulation, developmental promotion of organs, and lifespan regulation. Ae. aegypti has only one DNA methyltransferase gene (AaDnmt2) belonging to the cytosine methyltransferase family 2, which is the most deeply conserved and widely distributed gene among metazoans. Here, we show that in mosquitoes the introduced endosymbiont, Wolbachia, significantly suppresses expression of AaDnmt2, but dengue virus induces expression of AaDnmt2. Interestingly, we found that aae-miR-2940 microRNA, which is exclusively expressed in Wolbachia-infected mosquitoes, down-regulates the expression of AaDnmt2. Reversely, overexpression of AaDnmt2 in mosquito cells led to inhibition of Wolbachia replication, but significantly promoted replication of dengue virus, suggesting a causal link between this Wolbachia manipulation and the blocking of dengue replication in Wolbachia-infected mosquitoes. In addition, our findings provide an explanation for hypomethylation of the genome in Wolbachia-infected Ae. aegypti.

Bona fide evidence for natural vertical transmission of infectious salmon anemia virus in freshwater brood stocks of farmed Atlantic salmon (Salmo salar) in Southern Chile.

UNLABELLED: Infectious salmon anemia (ISA) is a severe disease that affects farmed Atlantic salmon (Salmo salar), causing outbreaks in seawater in most salmon-producing countries worldwide, with particular aggressiveness in southern Chile. The etiological agent of this disease is a virus belonging to the Orthomyxoviridae family, named infectious salmon anemia virus (ISAV). Although it has been suggested that this virus can be vertically transmitted, even in freshwater, there is a lack of compelling experimental evidence to confirm this. Here we demonstrate significant putative viral loads in the ovarian fluid as well as in the eggs of two brood stock female adult specimens that harbored the virus systemically but without clinical signs. The target virus corresponded to a highly polymorphic region 3 (HPR-3) variant, which is known to be virulent in seawater and responsible for recent and past outbreaks of this disease in Chile. Additionally, the virus recovered from the fluid as well as from the interior of the eggs was fully infective to a susceptible fish cell line. To our knowledge, this is the first robust evidence demonstrating mother-to-offspring vertical transmission of the infective virus on the one hand and the asymptomatic transmission of a virulent form of the virus in freshwater fish on the other hand. IMPORTANCE: The robustness of the data presented here will contribute to a better understanding of the biology of the virus but most importantly will constitute a key management tool in the control of an aggressive agent constantly threatening the sustainability of the global salmon industry.

Molecular profiles and pathogen-induced transcriptional responses of prawn B cell lymphoma-2 related ovarian killer protein (BOK).

In this study, we have reported a molecular characterization of the first B cell lymphoma-2 (BCL-2) related ovarian killer protein (BOK) from freshwater prawn Macrobrachium rosenbergii (Mr). BOK is a novel pro-apoptotic protein of the BCL-2 family that entails in mediating apoptosis to remove cancer cells. A cDNA sequence of MrBOK was identified from the prawn cDNA library and its full length was obtained by internal sequencing. The coding region of MrBOK yields a polypeptide of 291 amino acids. The analysis revealed that MrBOK contains a transmembrane helix at V(261)-L(283) and a putative BCL-2 family domain at V(144)-W(245). MrBOK also possessed four putative BCL-2 homology domains including BH1, BH2, BH3 and weak BH4. The BH3 contains 21 binding sites and among them five residues are highly conserved with the aligned BOK proteins. The homology analysis showed that MrBOK shared maximum similarity with the Caligus rogercresseyi BOK A. The topology of the phylogenetic tree was classified into nine sister groups which includes BOK, BAK, BAX, BAD, BCL-2, BCL-XL, NR13 and MCL members. The BOK protein group further sub-grouped into vertebrate and invertebrate BOK, wherein MrBOK located within insect monophyletic clad of invertebrate BOK. The secondary structural analysis showed that MrBOK contains 11 α-helices (52.2%) which are connected over random coils (47.7%). The 3D structure of MrBOK showed three central helices (α6, α7 and α8) which formed the core of the protein and are flanked on one side by α1, α2 and α3, and on the other side by α4, α5 and α11. MrBOK mRNA is expressed most abundantly (P < 0.05) in ovary compared to other tissues taken for analysis. Hence ovary was selected to study the possible roles of MrBOK mRNA regulation upon bacterial (Aeromonas hydrophila and Vibrio harveyi) and viral [white spot syndrome virus (WSSV) and M. rosenbergii nodovirus] infection. During bacterial and viral infection, the highest MrBOK mRNA transcription was varied at different time points. In bacterial infected ovary tissue, the highest mRNA expression was at 24 h post-infection, whereas in viral infection, the expression was highest at 48 h post-infection. Thus we can conclude that MrBOK functions as an apoptotic protein in intracellular programmed cell-death pathway to counteract the anti-apoptotic proteins released by bacterial and viral pathogens at the time of infection. This is the first study that emphasizes the importance of BOK during bacterial and viral infection in crustacean.

Prevalence of porcine circovirus-2 DNA-positive ovarian and uterine tissues in gilts culled due to reproductive disturbance in Thailand.

The present study aimed to determine the prevalence of porcine circovirus-2 (PCV-2) DNA-positive ovarian and uterine tissues in gilts culled due to reproductive disturbance in Thailand. Tissues (70 ovaries and 102 uteri) and serum (n = 102) samples from 102 gilts were included. PCV-2 DNA was detected by using polymerase chain reactions. The localisation of PCV-2 antigen was determined by immunohistochemistry, and PCV-2 antibody was evaluated by ELISA. PCV-2 DNA was detected in 30.0 % (21/70) of the ovaries and in 45.1 % (46/102) of the uteri. Age did not influence the frequency of PCV-2 DNA detection in these reproductive organs of gilts (P > 0.05). The prevalence of PCV-2 DNA-positive uterine tissue in gilts culled due to non-reproductive problems (20.0 %) was lower than gilts culled due to abortion (85.0 %), abnormal vaginal discharge (47.5 %) and anoestrus (53.5 %) (P < 0.05). The prevalence of PCV-2 DNA-positive uterine tissue in the gilts with high antibody titres (23.0 %) was lower than in gilts with low antibody titres (57.6 %) and seronegative gilts (64.5 %) (P < 0.05). PCV-2 immunostaining was detected in the endometrial cells, lymphocytes and macrophages of the uteri and in oocytes and granulosa cells of the ovaries. In conclusion, the detection of PCV-2 in the reproductive organs reveals an important potential impact of this virus on the reproductive apparatus in gilts.

A viral aphrodisiac in the cricket Gryllus texensis.

We identified the insect iridovirus IIV-6/CrIV as a pathogen of the cricket Gryllus texensis using electron microscopy (EM) and polymerase chain reaction (PCR) analysis. EM showed that the virus attacks the fat body, an organ important for protein production, immune function and lipid storage. During infection the fat body hypertrophied, but egg production withered, leaving the lateral oviducts empty of eggs; the females were effectively sterile. EM of the testis of infected males suggests that the testis was not invaded by the virus, although sperm taken from the spermatophores of infected males showed little or no motility. Nevertheless, males and females continued to mate when infected. In fact, infected males were quicker to court females than uninfected controls. The virus benefits from the continued sexual behaviour of its host; transmission studies show that the virus can be spread through sexual contact. Sickness behaviour, the adaptive reduction of feeding and sexual behaviour that is induced by an activated immune system, was absent in infected crickets. Total haemolymph protein was reduced, as was phenoloxidase activity, suggesting a reduction in immune protein production by the fat body. The evidence suggests that during IIV-6/CrIV infection, the immune signal(s) that induces sickness behaviour is absent. Curtailment of a host's sickness behaviour may be necessary for any pathogen that is spread by host sexual behaviour.